AAV vectors have shown promise for cystic fibrosis (CF) gene therapy in tissue culture, small animals, primates and CF patients, particularly with regard to safety and persistence. However, gene transfer in the airways for CF patients has been less efficient than was predicted from the in vitro studies and the animal data. There are a number of possible explanations for this. First, it is possible that the luminal surface of the lower airways of humans is deficient in receptors for AAV. In the case of adenovirus (Ad) vectors, vector tropism for the human nasal epithelium appears to be substantially lower than that observed for the bronchial epithelium of experimental animals. If an analogous discrepancy is present in the case of AAV, then alteration of the viral capsid may be required to enhance entry into human bronchial epithelial cells. Secondly, the exposure of AAV particles to free neutrophil elastase, extracellular leukocyte DNA, bacterial exoenzymes, or hyper- sulfated proteoglycans present in CF bronchial secretions may pose a particular obstacle to in vivo transduction in the lungs of CF patients. Thirdly, the small packaging capacity of AAV has led to the use of minimal endogenous AAV promotor elements for expression of CFTR within AAV vectors, which may be suboptimal for expression. Since the combination of larger promoter with full-length CFTR would produce vector cassettes which exceed the 5-kb packaging limit of AAV, the use of partially-truncated versions of the CFTR will need to be examined. These issues will be addressed in the following specific aims: 1. To assess the tropism of AAV vectors for the normal human bronchial epithelium in vivo. A new reporter system, based on fluorescent bronchoscopic detection of the Green Fluorescent Protein (GFP), will be used to assess gene transfer efficiently in normal human volunteers. 2. To determine whether there are specific barriers to AAV transduction which are greater in individuals with CF as compared with normal individuals. 3. To alter the tropism of AAV vectors to target specific receptors on the basolateral membrane in bronchial epithelial cells. In collaboration with Dr. Thomas Ferkol we will attempt to enhance AAV uptake and bypass mucosal barriers by coupling of vectors with antibodies directed against cellular receptors, such as the epidermal growth factor receptor (EGFR) and the polymeric immunoglobulin receptor (pIgR). To optimize the expression of CFTR from the AAV vector system, particularly in the context of using more active promoters to drive expression of CFTR minigenes. We anticipate that the completion of these aims will remove the remain obstacles to the development of a clinically useful AAV-based gene therapy for cystic fibrosis.